ES2636671T3 - Methods for risk prediction, diagnosis, prognosis of pulmonary disorders - Google Patents

Abstract

A method for determining if a subject has or is at risk of developing a pulmonary fibrotic condition, said method comprising detecting in a biological sample of said subject if a genome of said subject comprises the rs35705950 T allele and where the presence of the T allele indicates that said subject has or is at risk of developing a pulmonary fibrotic condition.

Description

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II. Mucins There are several gel-forming mucins that include, but are not limited to, MUC6, MUC2, MUC5AC, and MUC5B. These proteins are large filamentous and highly O-glycosylated.

III. Pulmonary Diseases Pulmonary diseases contemplated herein may include any pulmonary disorder, pulmonary fibrosis disease, interstitial lung disease, idiopathic interstitial pneumonia (NII), idiopathic pulmonary fibrosis, familial interstitial pneumonia (NIF), acute respiratory distress syndrome (ARDS) ), pulmonary scleroderma disease, Sarcoidosis, Beryllium disease, pulmonary disorder associated with rheumatoid arthritis, pulmonary disorder associated with vascular collagen, pulmonary disorder associated with cigarette smoke, Sjögren's syndrome, mixed connective tissue disease, non-specific interstitial pneumonia (NINE), etc.

Pulmonary fibrotic conditions, for example, interstitial lung diseases (ILD) are characterized by shortness of breath, chronic cough, fatigue and weakness, loss of appetite and rapid weight loss. Pulmonary fibrosis is commonly linked to interstitial lung diseases (for example, autoimmune disorders, viral infections or other microscopic lesions), but it can be idiopathic. Fibrosis involves the exchange of normal lung tissue with fibrotic tissue (scar tissue) that leads to reduced oxygen capacity.

Idiopathic interstitial pneumonia (NII) is a subset of diffuse interstitial lung diseases of unknown etiology (the term "idiopathic" indicates unknown origin). NIIs are characterized by the expansion of the interstitial compartment (i.e., that portion of the pulmonary parenchyma interspersed between the epithelial and endothelial basal membranes) with an infiltrate of inflammatory cells. The inflammatory infiltrate is sometimes accompanied by fibrosis, either in the form of abnormal collagen deposition or proliferation of fibroblasts capable of collagen synthesis.

Idiopathic Pulmonary Fibrosis (IPF) occurs in thousands of people worldwide with a doubling in prevalence over the past 10 years. The appearance of IPF occurs around 50 to 70 years of age and begins with progressive difficulty breathing and hypoxemia. The average survival of IPF is around 3-5 years and is up to date intractable. The etiology and pathogenesis of the condition is not well understood. Approximately 5-20 percent of all IPF cases have a family history and inheritance appears to be autosomal dominant.

Additional fibrotic pulmonary diseases include acute interstitial pneumonia (AIP), Respiratory bronchiolitis associated with Pulmonary Interstitial Disease (RBILD), Descamative Interstitial Pneumonia (NID), Non-Specific Interstitial Pneumonia (NINE), Obligatory Bronchiolitis, with Organizational Pneumonia (BONO).

AIP is a rapidly progressive and histologically distinct form of interstitial pneumonia. The pathological pattern is a form of diffuse alveolar damage (DAD) organization that is also found in acute respiratory distress syndrome (ARDS) and other acute interstitial pneumonia of known causes (see Clinical Atlas of Interstitial Lung Disease (2006 ed.) pp61-63).

RBILD is characterized by inflammatory lesions of the respiratory bronchioles in cigarette smokers. The histological aspect of RBILD is characterized by the accumulation of pigmented macrophages within the respiratory bronchioles and surrounding air spaces, variably, peribronchial fibrotic septal thickening, and associated minimal mural inflammation (see Wells et al. (2003) Sem Respir. Crit Care Med. Vol. 24).

NID is a rare interstitial lung disease characterized by the accumulation of macrophages in large quantities in the alveolar spaces associated with interstitial inflammation and / or fibrosis. Macrophages often contain light brown pigment. Lymphoid nodules are common, as well as a dispersed but distinct eosinophil infiltrate. NID is more common in smokers (see Tazelaar et al. (Sep. 21, 2010) Histopathology).

NINE is pathologically characterized by uniform interstitial inflammation and fibrosis that appear for a short period of time. NINE differs from other interstitial lung diseases in that it has a generally good prognosis. In addition, the temporal uniformity of the parenchymal changes observed in NINE contrasts sharply with the temporal heterogeneity of usual interstitial pneumonia (see Coche et al. (2001) Brit J Radiol 74: 189).

BONUS, unlike NINE, can be fatal within days of the first acute symptoms. It is characterized by the rapid onset of acute respiratory distress syndrome; Therefore, clinically, rapidly progressive BONUS can be indistinguishable from acute interstitial pneumonia. Histological features include clusters of mononuclear inflammatory cells that form granulation tissue and plug the distal airways and alveolar spaces. These granulation tissue plugs may form polyps that migrate within the alveolar ducts or may be focally attached to the wall (see White & Ruth-Saad (2007) Crit. Care Nurse 27:53).

Additional details about the features and therapies available for these diseases can be found, for example, on the website of the American Lung Association at lungusa.org/lung-disease/pulmonary-fibrosis.

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Diagnostic indicators of pulmonary disorders include biopsy (for example, VATS or surgical lung biopsy), high-resolution computed tomography (HRCT) or breathing metrics, such as forced expiratory volume (FEV1), vital capacity (CV), capacity forced vital (CVF) and VEF1 / CVF.

Additional disorders associated with the expression of MUC5B and / or SNPs associated with MUC5B (for example, SNP rs35705950) may include, but are not limited to, mucous secretion disorders, cancers (eg, ovarian, breast, lung, pancreatic, etc.), eye disease, colitis and cirrhosis of the liver.

IV.

 Diagnostic and Prognostic Methods Methods to detect and identify nucleic acids and proteins and interactions between such molecules involve techniques of conventional molecular biology, microbiology and recombinant DNA within the skill of the art. Such techniques are fully explained in the literature (see, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Animal Cell Culture, RI Freshney, ed., 1986).

TO.

 Biological samples For the detection of a genetic variant using genomic DNA, a biological sample of almost any tissue can be obtained. One skilled in the art will understand that a blood sample or a cheek swab is expected to carry the same genetic sequence information as a lung cell. For the detection of a given level of expression, samples of lung tissue and other biological fluids are typically used.

Biological samples may include a sample of pulmonary mucosa or biological fluid such as blood or blood components (plasma, serum), sputum, mucus, urine, saliva, etc.

A sample of pulmonary mucosa can be obtained using methods known in the art, for example, a bronchial epithelial brush or exhaled air condensate. Additional methods include bronchial biopsy, bronchial lavage, bronchoalveolar lavage, complete lung lavage, transendoscopic biopsy, catheter after laryngoscopy and transtracheal lavage. A review of commonly used techniques, including comparisons and safety issues, is provided in Busse et al. (2005) Am J Respir Crit Care Med 172: 807-816.

For washing techniques, a bronchoscope can be inserted to the desired level of the airways. A small volume of sterile, physiologically acceptable fluid (for example, regulated saline) is released, and immediately aspirated. The washing material contains mucosal cells and upper epithelia (Riise et al. (1996) Eur Resp J 9: 1665).

For the use of a bronchial epithelial brush, a sterile, non-irritating cytology brush can be used (for example, nylon). Multiple brushings can be taken to ensure representative sampling. The brush is then agitated in physiologically acceptable fluid, and the cells and debris are separated using routine methods (Riise et al. (1992) Eur Resp J 5: 382).

Cellular components can be isolated using methods known in the art, for example, centrifugation. Similarly, subcellular components (eg, exosomes or vesicles) can be isolated using known methods or commercial separation products (available from BioCat, System Bio, Bioscientific, etc.). An exemplary method is described, for example, by Thery et al. (2006) Current Prot. Cell Biol.

B. Detection of genetic variants The inventors have found that genetic variations in mucin genes are associated with lung diseases. These genetic variations can be found anywhere in the gene, for example, in regulatory regions, introns, or exons. Relevant genetic variations can also be found in intergene regions, for example, in sequences between mucin genes. Insertions, substitutions and deletions are included in the genetic variants. Single nucleotide polymorphisms (SNPs) are exemplary genetic variants.

In particular, 14 independent SNPs are associated with pulmonary disorders (for example, NIF or FPI). The studies described herein demonstrate that the presence of one or more of these SNPs associated with MUC5B can lead to predisposition to lung disease. In addition, if present, some of these SNPs are related to a transcription factor binding site. The transcription factor binding site can effect the modulation of MUC5B expression, for example loss of E2F3, and the generation of HOXA9 and PAX-2.

The invention therefore provides methods for assessing the presence or absence of SNPs in a sample of a subject suspected of having or developing a lung disorder (for example, due to family history). In certain embodiments of the disclosure, one or more SNPs are screened in one or more samples of a subject. SNPs can be associated with one or more genes, for example, one or more MUC genes or other genes associated with mucous secretion. An SNP associated with the MUC gene is associated with MUC5B and / or another MUC gene, such as MUC5AC or MUC1. SNPs contemplated for diagnosis, treatment or prognosis may include SNPs found within a MUC gene and / or within a regulatory or promoter region associated with a MUC gene. For example, one or more SNPs may include, but are not limited to, the detection of the MUC5B SNPs shown in Table 4 (SEQ ID Nos: 20-53), for example, SNPs.

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Mucolytic drugs are those that decrease the viscosity of mucus, either by depolymerization of mucin glycoproteins or by depolymerization of polymeric networks of DNA and F-actin. The first mode of action may be particularly useful for treating excess MUC5B. Exemplary mucolytics include N-acetylcysteine, Nacysteline, erdostein, dornase alfa, thymosin beta4, dextran, pulmozyme, heparin and bronchiotol (inhaled mannose).

Mucorregulators are those agents that regulate mucus secretion, or interfere with the DNA / F-actin network. Examples of mucorregulators include, for example, carbocysteine, anticholinergic agents, glucocorticoids and macrolide antibiotics.

Mucokinetic agents increase mucus removal by acting on the ciliary lining of the airways. Exemplary mucokinetic agents include, for example, bronchodilators, surfactants and ambroxol.

Expectorants are agents that induce the discharge of mucus from the airways or respiratory tract. Some examples include hypertonic saline, guaifenesin, dornase / pulmozyme and bronchiotol (inhaled mannose).

The treatment of lung disease, such as the agents described above, can be used alone, sequentially, or in combination according to the methods described herein. A lung disease treatment can be used in combination with a more targeted inhibitor of MUC5B expression.

B. MUC5B antagonists The results described herein indicate that elevated expression of the MUC5B gene is associated with lung disease. The disclosure therefore includes methods and compositions to inhibit the expression, secretion and / or activity of MUC5B. Exemplary inhibitors include siRNA and antisense, pRNA (promoter-associated RNA, see, for example, Schmitz et al. (2010) Genes Dev. 24: 2264-69), MUC5B specific antibodies and fragments thereof, and specific aptamers of MUC5B. The activity of MUC5B can be inhibited or the elimination of MUC5B can be increased, for example, using mucolytic agents, glycosylation inhibitors, or protein secretion inhibitors. The terms "inhibitor" and "antagonist" and similar terms are used synonymously herein.

Therefore, a nucleotide sequence that specifically interferes with the expression of the MUC5B gene at the level of transcription or translation can be used to treat or prevent lung disease. This approach can use, for example, siRNA and / or antisense oligonucleotides to block the transcription or translation of a specific mRNA (eg, a genetic variant RNA), either by inducing degradation of the mRNA with an siRNA or masking the mRNA with an antisense nucleic acid. The siRNA or antisense construct may not significantly block the expression of other mucin genes.

The double stranded siRNA corresponding to the MUC5B gene can be used to silence transcription and / or translation by inducing degradation of MUC5B mRNA transcripts, and therefore treating or preventing lung disease (e.g., lung disease associated with MUC5B of genetic variant). The siRNA is typically about 5 to about 100 nucleotides in length, more typically about 10 to about 50 nucleotides in length, most typically about 15 to about 30 nucleotides in length. SiRNA molecules and the methods to generate them are described in, for example, Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498; WO 00/44895; WO 01/36646; WO 99/32619; WO 00/01846; WO 01/29058; WO 99/07409; and WO 00/44914. A DNA molecule that transcribes siRNA or siRNA (for example, as a hairpin duplex) also provides RNAi. DNA molecules for transcribing siRNA are described in US Pat. No. 6,573,099, and in U.S. Patent Application Publication Nos 2002/0160393 and 2003/0027783, and Tuschl and Borkhardt, Molecular Interventions, 2: 158 (2002). For example, dsRNA oligonucleotides that specifically hybridize with the MUC5B nucleic acid sequences described herein can be used in the methods. A decrease in the severity of pulmonary disease symptoms can be used compared to the symptoms detected in the absence of interfering RNA to monitor the effectiveness of siRNA.

Antisense oligonucleotides that specifically hybridize with nucleic acid sequences encoding MUC5B polypeptides can also be used to silence transcription and / or translation, and therefore treat or prevent lung disease. For example, antisense oligonucleotides that specifically hybridize with a polynucleotide sequence of MUC5B can be used. A decrease in the severity of lung disease symptoms can be used compared to the symptoms detected in the absence of antisense nucleic acids to monitor the efficacy of antisense nucleic acids.

Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (see, for example, Weintraub, Scientific American, 262: 40 (1990)). Typically, synthetic antisense oligonucleotides are generally between 15 and 25 bases in length. Antisense nucleic acids may comprise naturally occurring nucleotides or modified nucleotides such as, for example, phosphorothioate, methylphosphonate, and anomeric sugar phosphate modified main chain nucleotides.

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In the cell, antisense nucleic acids hybridize with the corresponding mRNA, forming a double chain molecule. Antisense nucleic acids interfere with the translation of mRNA, since the cell will not translate an mRNA that is double stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target mutant nucleotide-producing cell. The use of antisense methods to inhibit in vitro translation of genes is well known in the art (Marcus-Sakura, Anal. Biochem., 172: 289, (1988)). Less commonly, antisense molecules that bind directly to DNA can be used.

The siRNA and antisense can be delivered to the subject using any means known in the art, including by injection, inhalation or oral ingestion. Another suitable delivery system is a colloidal dispersion system such as, for example, complexes of macromolecules, nanocapsules, microspheres, globules, and lipid-based systems that include oil-in-water emulsions, micelles, mixed micelles and liposomes. The preferred colloidal system is a liposome. Liposomes are artificial membrane vesicles that are useful as in vitro and in vivo delivery vehicles. Nucleic acids, including RNA and DNA within the liposomes and be supplied to the cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981). Liposomes can be directed to specific cell types or tissues using any means known in the art.

The disclosure also provides antibodies that specifically bind to the MUC5B protein. Such antibodies can be used to sequester the secreted MUC5B, for example, to prevent gel-forming activity and the formation of excess mucus.

An antibody that specifically detects MUC5B, and not other mucin proteins, can be isolated using the standard techniques described herein. Protein sequences for MUC5B in a number of species, for example, humans, nonhuman primates, rats, dogs, cats, horses, cattle, etc., are publicly available.

Monoclonal antibodies are obtained by various techniques familiar to those skilled in the art. Briefly, the spleen cells of an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, for example, Kohler & Milstein, Eur. J. Immunol. 6: 511-519 (1976)) . Alternative methods of immortalization include transformation with Epstein-Barr virus, oncogenes or retroviruses, or other methods well known in the art. Colonies that arise from individual immortalized cells are screened for the production of antibodies of the specificity and affinity desired for the antigen, and the production of monoclonal antibodies produced by such cells can be increased by various techniques, including injection into the peritoneal cavity. of a vertebrate host. Alternatively, DNA sequences encoding a monoclonal antibody or a binding fragment thereof can be isolated by screening a human B cell DNA library according to the general protocol indicated by Huse et al., Science 246: 1275-1281 (1989) .

Monoclonal antibodies are collected and titrated against MUC5B in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Monoclonal antibodies will usually bind with a Kd of at least about 0.1mM, and more usually of at least about 1 µM, and can often be designed to bind with a Kd of 1 nM or less.

Immunoglobulins, including MUC5B-binding fragments and derivatives thereof, can be easily produced by a variety of recombinant DNA techniques, including by expression in transfected cells (eg, immortalized eukaryotic cells, such as myeloma cells or hybridoma) or in mice, rats, rabbits, or another vertebrate capable of producing antibodies by well known methods. Suitable source cells for DNA sequences and host cells for immunoglobulin expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection (Catalog of Cell Lines and Hybridomas, Fifth Edition (1985) Rockville , Md).

The antibody is a humanized antibody, i.e., an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for example, by conserving non-human CDR regions that are specific for MUC5B, and replacing the remaining parts of the antibody with their human counterparts. See, for example, Morrison et al., PNAS USA, 81: 6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44: 65-92 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988); Padlan, Molec. Immun., 28: 489-498 (1991); Padlan, Molec. Immun., 31 (3): 169-217 (1994). Techniques for humanizing antibodies are well known in the art and are described in, for example, US Pat. US 4,816,567; 5,530,101; 5,859,205; 5,585,089; 5,693,761; 5,693,762; 5,777,085; 6,180,370; 6,210,671; and 6,329,511; WO 87/02671; European Patent Application 0173494; Jones et al. (1986) Nature 321: 522; and Verhoyen et al. (1988) Science 239: 1534. Humanized antibodies are further described in, for example, Winter and Milstein (1991) Nature 349: 293. For example, polynucleotides comprising a first sequence encoding the humanized immunoglobulin structure regions and a second set of sequences encoding the desired immunoglobulin complementarity determining regions can be produced synthetically or by combining appropriate cDNA or genomic DNA segments. Human constant region DNA sequences can be isolated according to well known procedures from a variety of human cells.

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peptidyl phosphonates (Campbell et al., J. Org. Chem. 59: 658 (1994)), nucleic acid libraries (ee Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, for example, the Patent No. 5,539,083), antibody libraries (see, for example, Vaughn et al., Nature Biotechnology, 14 (3): 309-314 (1996) and PCT / US96 / 10287), carbohydrate libraries (see, for example, Liang et al., Science, 274: 1520-1522 (1996) and US Patent 5,593,853), libraries of small organic molecules (see, for example, benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134, compounds Morpholino, U.S. Patent 5,506,337, benzodiazepines, and U.S. Patent No. 5,288,514).

D. Pharmaceutical compositions The compositions described herein may be administered by any means known in the art. For example, the compositions may include administration to a subject intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostically, intrapleurally, intratracheally, intranasally, intravitrally, intravaginally, intarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneous, subconjunctive, intravesicular, mucosal, intrapericardial, intraumbilical, intraocular, oral, local, by inhalation, by injection, by infusion, by continuous infusion, by localized infusion, through a catheter, by washing, in a cream, or in a lipid composition. Administration may be local, for example, to the pulmonary mucosa, or systemic.

Solutions of the active compounds as a free base or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant, such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under normal conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

The pharmaceutical compositions can be supplied by intranasal or inhalable solutions or sprays, aerosols or inhalers. Nasal solutions may be aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions can be prepared so that they are similar in many ways to nasal secretions. Therefore, aqueous nasal solutions are usually isotonic and slightly regulated to maintain a pH of 5.5 to 6.5. In addition, if required, antimicrobial preservatives, similar to those used in ophthalmic preparations, and appropriate drug stabilizers may be included in the formulation. Various commercial nasal preparations are known and may include, for example, antibiotics and antihistamines.

Oral formulations may include excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. The oral pharmaceutical compositions may comprise an inert diluent or assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly into the diet food. For oral therapeutic administration, the active compounds can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, pills and the like. Such compositions and preparations must contain at least 0.1% active compound. The percentage of the compositions and preparations can, of course, be varied and may conveniently be between about 2 to about 75% of the weight of the unit, or preferably between 25-60%. The amount of active compounds in such compositions is such that a suitable dosage can be obtained.

For parenteral administration in an aqueous solution, for example, the solution must be adequately regulated and the liquid diluent first made isotonic with sufficient saline or glucose. Aqueous solutions, in particular, sterile aqueous media, are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. For example, a dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed infusion site.

Sterile injectable solutions can be prepared by incorporating the active compounds or constructs in the required amount in the appropriate solvent followed by filtration sterilization. Generally, the dispersions are prepared by incorporating the various sterilized active ingredients in a sterile vehicle containing the basic dispersion medium. Vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients, can be used to prepare sterile powders for reconstitution of sterile injectable solutions. The preparation of more, or highly, concentrated solutions for direct injection is also contemplated. DMSO can be used as a solvent for extremely fast penetration, providing high concentrations of active agents to a small area.

E. Treatment regimens The disclosure provides methods for treating, preventing and / or improving a lung disorder in a subject in need thereof, optionally based on the diagnostic and prediction methods described herein. The course of treatment is best determined on an individual basis depending on the particular characteristics of the subject and the type of treatment selected. The treatment, such as those described herein, can be administered at

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subject daily, twice a day, biweekly, monthly, or on any applicable basis that is therapeutically effective. The treatment may be administered alone or in combination with any other treatment described herein or known in the art. The additional treatment can be administered simultaneously with the first treatment, at a different time, or in a completely different therapeutic program (for example, the first treatment may be daily, while the additional treatment is weekly).

The administration of a composition to improve lung disease, for example, by treating the elevated expression of the MUC5B gene, can be a systemic or localized administration. For example, treatment of a subject who has a pulmonary disorder may include the administration of an inhalable or intranasal form of anti-MUC5B agent (MUC5B antagonist) daily or an otherwise regular schedule. Treatment may only be on a basis as needed, for example, after the onset of symptoms of lung disease.

SAW. Equipment The disclosure provides equipment for the detection of markers of lung disease in a subject. The equipment can be for personal use or provided to medical professionals. The team can be a team to diagnose or predict a lung disorder, or to monitor the progression of the disease or the effectiveness of the treatment.

The kit may include components for evaluating the expression of the MUC5B gene comprising, for example, a nucleic acid capable of detecting MUC5B RNA or an optionally labeled MUC5B protein binding agent. An expert will appreciate that the expression of the MUC5B gene can be determined by measuring the MUC5B RNA or protein. The equipment may also include test vessels (tubes), regulatory solutions, or enzymes necessary to carry out the detection test.

The kit may include components to determine whether the subject's genome carries a gene variant MUC5B gene, for example, a nucleic acid that specifically hybridizes with a gene variant MUC5B gene sequence. Other components in a kit may include DNA sequencing assay components, Taqman ® genotyping assay components, Meta-analysis, one or more detection system (s), one or more control samples or a combination thereof. The teams may also include one or more agents where at least one of the agents is able to associate with the SNP rs35705950.

The team may include components to examine more than one marker of lung disease. For example, the equipment may include marker detection agents, such as primers or specific marker probes linked to an airship arrangement. Exemplary markers include SNPs in the MUC5B genes, or genetic variants in other genes, for example, Surfactant Protein A2, Surfactant Protein B, Surfactant Protein C, TERC, TERT, IL-1RN, IL-1α, It-1β, TNF , Lymphotoxin α, TNF-RII, IL-10, IL-6, IL-I2, IFNγ, TGFβ, CR1, ACE, IL-8, CXCR1 or CXCR2. The level of expression of the markers can be detected instead of or in addition to the genetic sequence. In this case, useful lung disease markers with aberrant expression include: Surfactant Protein A, Surfactant Protein D, KL6 / MUC1, CC16, CK-19, Ca 19-9, SLX, MCP-1, MIP-1a, ITAC, glutathione, procollagen peptide type III, sIL-2R, ACE, neopterin, beta-glucuronidase, LDH, CCL-18, CCL-2, CXCL12, MMP7, and osteopontin. Additional lung disease markers may include the other MUC genes, for example, MUC2, MUC5AC, and MUC6.

The equipment will generally include at least one vial, test tube, flask, bottle, syringe or other container means, in which the test agent can be reacted or divided into aliquots properly. The equipment may also include components for comparing the results such as a suitable control sample, for example a positive and / or negative control. The equipment may also include a collection device to collect and / or save the subject's sample. The collection device may include a sterile swab or needle (for collecting blood), and / or a sterile tube (for example, for storing the swab or a sample of body fluid).

VII. Examples

Example 1: Sequencing of lung mucins, gel-forming and association with the disease Study populations: subjects with NIF or FPI were identified and phenotyped. The diagnosis of NII was established according to conventional criteria. Eligible subjects were at least 38 years old and had symptoms of NII for at least 3 months. A high resolution computed tomography (HRCT) scan was required to show the defined or probable NII according to the predefined criteria, and a surgical lung biopsy was obtained in 46% of the affected subjects. IPF families were defined by the presence of two or more cases of defined or probable NII within three grades, with at least one case of NII established as defined / probable IPF. Exclusion criteria included significant exposure to known fibrogenic agents or an alternative etiology for ILD. Control subjects were acquired for genetic analysis (Fig. 1).

Link analysis: a wide genome link screening was completed in 82 multiplex families using a DeCode link panel consisting of a total of 884 markers with an average inter-marker distance of 4.2 CM. Multipoint non-parametric link analysis was performed using Merlin, previously described. The LOD scores of Kong and Cos were calculated using Spares statistics in an exponential model; Support intervals were determined using the LOD score down method.

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Fine mapping of chromosome 11: To interrogate the region linked at the p-end of chromosome 11 (8.4 Mb linked by rs702966 and rs1136966), fine mapping was performed genotyping 306 tagging SNPs in 145 unrelated cases of NIF, 152 cases of IPF , and 233 Caucasian controls. Association tests comparing NIF cases and FPI cases with controls were calculated under an additive model for the minor allele.

Resequencing of MUC2 and MUC5AC: primer pairs were designed to generate overlapping amplicons for resequencing the proximal promoter and most of the exons of MUC2 and MUC5AC over masked sequences for repetitive elements, SNPs, and homology with other regions of the genome.

Genetic screening of gel-forming mucins, expressed in the lung: a case control association study was conducted in an independent population of NIF (N = 83), sporadic IPF (N = 492), and control subjects (N = 322) (Table 2) using labeling and other SNPs located through the mucin genes expressed in the lung, gel-forming on chromosome 11. 175 SNPs were successfully genotyped using the Sequenom iPlex assays, and haploview was used to test the SNP for allelic association with NIF and FPI. For those SNPs that remained significant after the Bonferroni correction, the likelihood ratios were estimated under an additive model for the rare allele after adjustment for age and gender through logistic regression. Chi-square goodness of fit tests were calculated to assess the evidence of disease model explanations for genotypic deviations of Hardy Weinberg Balance (HWE) between cases. For the most highly associated SNP, the link modeling and association in pedigrees was used to test whether, in the original binding families, the SNP was linked to the disease locus, was in imbalance of liaison with the disease locus, and could explain the link signal.

Strong evidence was produced for the link based on the 82 families of NIF on chromosome 11 where the maximum multipoint LOD score was 3.3 (p = 0.00004, D11S1318; Fig. 3). The 1-LOD support interval for this linked region was linked by markers D11S4046 and D11S1760, which cover 3.4 Mb. Since D11S4046 was the most typified telomeric marker, the region of interest was inclusive of the p-end of chromosome 11 Within the larger 8.4 Mb region, 306 tagging SNPs were selected for fine mapping in a case control association analysis (145 cases of NIF, 152 cases of IPF, and 233 controls. Association tests allelic revealed 7 SNPs within the mucin 2 (MUC2) gene significantly associated with either NIF or FPI.MUC2 is contained in a genomic region that hosts 4 gel-forming mucin genes (centromere telomere: MUC6, MUC2, MUC5AC, and MUC5B ) Although recombination hot spots are located between MUC6 and MUC2, and within the proximal portion of MUC5B, markers within MUC2 and MUC5AC exhibit strong link imbalance (LD) 17. Therefore, MUC2 and MUC5AC were selected for resequencing using oligonucleotide primers. Resequencing analysis identified 330 genetic variants in MUC2 and 195 genetic variants in MUC5AC. Allelic association tests between these genetic variants and the disease status produced 7 independent SNPs in both MUC2 and MUC5AC significantly associated with the disease status of either NIF or FPI.

The inventors designed a genetic screening for common genetic variation across the genomic region that contains the 3 gel-forming mucin genes expressed in the lung (MUC2, MUC5AC, and MUC5B) in an independent population of subjects with NII (NIF = 83 and FPI = 492) and controls (n = 322) (Fig. 1, Table 2). It was observed that 19 independent SNPs were significantly associated by allelic testing with either or both of NIF or FPI after Bonferroni correction for multiple comparisons (Table 1). Of these 19 SNPs, 6 occurred in MUC2, one in the intergenic region of MUC2-MUC5AC, 4 in MUC5AC, 3 in the intergenic region of MUC5AC-MUC5B, and 5 in the putative MUC5B promoter, within 4kb of site 18, 19 of the start of transcription of MUC5B (Table 1).

Of importance, it was found that an SNP in the putative promoter of MUC5B, 3kb upstream of the transcription initiation site (rs35705950) has the most important effect on both NIF and FPI. The minor allele of this SNP was present at a frequency of 33.8% in NIF cases, 37.5% in FPI cases, and 9.1% among controls (allelic association; NIF P = 1.2x10 -15, FPI P = 2.5x10-37). In particular, the genotype frequencies for rs35705950 were consistent with HWE in the controls, but not among the cases of IPF (P = 6.0x10-11) and almost so among the cases of NIF (P = 0.11). Comparing the genotype frequencies observed in cases and controls with those expected if rs35705950 is a real risk locus, the data demonstrate that these genotype frequencies are consistent with an additive genotypic effect on the risk of disease conferred by rs35705950 (P = 0.88 and P = 0.77, respectively for NIF and FPI to reject the additive effect). In addition, the frequency of the disease allele and penetrance estimates suggest a similar disease model for both NIF and FPI. The probability ratio for the disease for heterozygous and homozygous subjects for the rare allele of this SNP was 6.8 (95% CI 3.9-12.0) and 20.8 (95% CI 3.8-113, 7) for NIF, and 9.0 (95% CI 6.2-13.1) and 21.8 (95% CI 5.1-93.5) for IPF (Table 1). To ensure that this SNP was not labeling another SNP in the MUC5B promoter region, the 4kb region was resequenced upstream of the MUC5B transcription initiation site in 48 cases of IPF and 48 controls (Table 3). It was observed that 34 genetic variants but none had an LD value of R2 in pairs with rs35705950 above 0.2 (Table 4). Finally, among the original link families, it was found that rs35705950 was both linked to (P = 0.04) and in link imbalance with (P = 1.5x10-9) the disease locus. Although there is some evidence of other linked variants in the region (P = 0.054), these results verify the relevance of this SNP to the disease in these families.

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Example 2: rs35705950 single nucleotide polymorphism results in increased expression of the MUC5B gene

The wild-type G allele of SNP rs35705950 is conserved among primate species. The SNP is directly 5 ’to a highly conserved region among vertebrate species, and is in the middle of the sequence that is predicted to be involved in the regulation of the MUC5B gene. A bioinformatic analysis of the effect of SNP rs35705950 predicts an alteration of an E2F binding site and the creation of at least two new binding sites (for example, HOX9 and PAX2).

Based on these analyzes, the effect of rs35705950 on the expression of the MUC5B gene was examined. In lung tissue of 33 subjects with IPF and 47 unaffected subjects, quantitative RT-PCR revealed that MUC5B gene expression was over regulated 14.1 times among subjects with IPF compared to unaffected subjects (P = 0 , 0001, Fig. 4A). A 37.4 fold increase in MUC5B expression was observed among unaffected subjects who carry at least one copy of the variant allele compared to homozygous wild type subjects (P = 0.0003, Fig. 4B). In contrast, no significant difference in the expression of the MUC5B gene was observed between subjects with IPF with at least one variant allele of rs35705950 (Fig. 4C). Smoking, a potential confounding factor for the expression of MUC5B, seemed to have little effect on the association between the rs35705950 variant allele and the expression of MUC5B between either subjects unaffected or affected by IPF (Figs. 4B and 4C).

Immunohistochemical staining of MUC5B in lung tissue showed cytoplasmic staining in secretory columnar cells of the bronchial tubes and larger proximal bronchioles (> 200 µm) in cases of IPF and controls (Fig. 5A). In subjects with IPF, regions of dense accumulation of MUC5B were observed in microscopic screening areas and involved patched staining of the metaplastic epithelial lining of honeycomb cysts (Fig. 5B), as well as mucosal plugs within the cysts (Fig. . 5C). There were no obvious differences in the staining characteristics of MUC5B in cases of IPF with the polymorphism of the MUC5B promoter.

Subjects with IPF have significantly more expression of the MUC5B lung gene than controls, and the MUC5B protein is expressed in pathological lesions of IPF. The present results show that the risk of developing NIF or FPI is substantially correlated with the promoter polymorphism re35705950, which causes increased MUC5B expression. Together, the data show that the expression of MUC5B in the lung plays a role in the pathogenesis of lung disease.

Based on the relationship between the SNP and excess production of MUC5B, too much MUC5B can impair the mucosal host's defense against excessive lung injury from inhaled substances, and over time, lead to the development of NII. In addition to the MUC5B promoter SNP, common exposures and basic biological processes can influence either the expression or elimination of MUC5B. For example, the expression of MUC5B can be increased in the lung by cigarette smoke, acrolein, oxidative stress, IL-6, IL-8, IL-13, IL-17, 17β-estradiol, extracellular nucleotides, or epigenetic changes that alter DNA methylation or chromatin structure. In addition, the elimination of pulmonary mucus is dependent on effective ciliary movement, adequate hydration of the pericillary fluid layer and an intact cough. Regardless of the cause, the present results indicate that the excess of MUC5B can compromise the defense of the mucosal host, reduce the removal of the lung from inhaled particles, dissolved chemicals and microorganisms. Given the importance of environmental exposures, such as asbestos, silica and other contaminants in the development of other forms of interstitial lung disease, it is logical to speculate that common inhaled particles, such as those associated with cigarette smoke or air pollution It could lead to or contribute to exaggerated interstitial injury in individuals who have defects in the defense of the mucosal host.

In addition, excess MUC5B in the respiratory bronchioles may interfere with alveolar repair. The alveolar lesion can lead to the collapse of the bronchoalveolar units and this focal pulmonary lesion is repaired through the re-epithelialization of the alveolus by the alveolar epithelial cells of type II. Therefore, MUC5B can prevent alveolar repair either by interfering with the interaction between type II alveolar epithelial cells and the underlying matrix, or by interfering with the surface tension properties of the surfactant. Any failure to re-epithelize the basal alveolus plate or the suboptimal surfactant biology could increase the ongoing collapse and fibrosis of adjacent bronchoalveolar units, and ultimately result in NII.

IPF lesions are spatially heterogeneous, suggesting that IPF is multifocal, originating in individual bronchoalveolar units. Since the rs35705950 SNP occurs in the promoter region of MUC5B and is predicted to alter the binding sites of the transcription factor, ectopic production of MUC5B in cells or locations that cause injury to the bronchoalveolar unit may be a causative agent. Non-screened genetic variants (especially in the inaccessible repetitive mucin regions) may be in imbalance of binding to the SNP of the MUC5B promoter and affect the function of other lung mucins.

The present observations provide a novel clinical approach to pulmonary disorders such as NII. The invocation of the mucins of the airways secreted in the pathogenesis of pulmonary fibrosis suggests that the airspace plays a role in the pathogenesis of NII. Although the SNP (rs35705950) in the MUC5B promoter can be used to identify individuals at risk of developing NII, the observation that mucin biology is

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